Method for detection of vitamin d metabolites

ABSTRACT

The present invention relates to a method for measuring the amount of vitamin D metabolite in a sample of plasma or serum, comprising the step of (a) adding to a serum or plasma sample a non-competitive displacement agent to effect separation of any vitamin D metabolite in the sample form protein to which it is bound, such that any vitamin D metabolite may be detected and/or measured; and (b) detecting or measuring the amount of vitamin D metabolite in the sample of (a). Such methods are useful in determining the vitamin D status of the subject, for example in the diagnosis of disease. Also provide are agents and kits for use in performing the methods of the invention.

[0001] The present invention relates to a method for measuring theamount of a vitamin D metabolite present in a sample of plasma or serum,and in particular to a method which enables measurement of the amount ofa vitamin D metabolite in a sample of plasma or serum without the needfor prior extraction from the sample of vitamin D metabolite bindingproteins. The present invention also relates to methods for determiningthe vitamin D status of a subject, for use in the diagnosis of disease,and to agents and kits for use in performing the methods of theinvention.

[0002] Vitamin D status is an indication of the level of circulatingvitamin D or vitamin D metabolites in a subject, and is an importantfactor in the cause of a number of diseases including rickets inchildren and osteomalcia in adults. It is also useful in identifying theunderlying causes of hypercalceamia and hypocalceamia. Vitamin D and itsmetabolism are the subject of great clinical interest, and in line withthis there has been a steady increase in the effort towards improvingmethods for the measurement of vitamin D and its metabolites in bodyfluids.

[0003] Vitamin D is available to man as either vitamin D₂ or vitamin D₃.Vitamin D₂ is produced outside the body by irradiation of ergosterolfrom yeast and fungi, and is found in man when taken in the form offortified foods or pharmaceutical preparations. Vitamin D₃, on the otherhand, is formed in animals from 7-dehydrocholesterol upon exposure to uvlight. In man, this reaction occurs in the skin. Vitamin D₃ is alsoavailable in the diet, for example from fish liver oils. Vitamin D, inthe form of vitamin D₂ or D₃, is rapidly converted to the circulatingmetabolite, 25-hydroxy vitamin D, which is found outside cells, tightlybound to circulating vitamin D binding protein (DBP). Due to the rapidconversion of vitamin D to its' metabolite, measurement of vitamin Ddoes not give a useful indication of the vitamin D status of a subject.Other metabolites of vitamin D, such as 1α,25-dihydroxy vitamin D,circulate at a concentration 1000 times lower than non-1α-hydroxylatedmetabolites such as 25-hydroxy vitamin D, and so do not contributesignificantly to the estimation of total circulating vitamin Dmetabolite. For this reason, the 1α-hydroxylated metabolites do notprovide a direct or useful indication of vitamin D status. 25-hydroxyvitamin D is the metabolite with the highest serum concentration, and iseasy to measure. It has therefore become the most common marker ofvitamin D status in a subject.

[0004] The established methods for measurement of 25-hydroxy vitamin Dprovide a result which includes contributions from other vitamin Dhydroxylated metabolites, principally 24,25-dihydroxy vitamin D but also25,26-dihydroxy vitamin D and other metabolites. These otherhydroxylated metabolites contribute 1 to 7% of the measure of totalcirculating vitamin D metabolites, the remainder being 25-hydroxyvitamin D. Whilst for many applications, including research, it issufficient to measure the total amount of circulating vitamin Dmetabolites, for other applications separation of these metabolites isrequired to allow specific measurement of 25-hydroxy vitamin D. It isgenerally accepted that measurement of 25-hydroxy vitamin D levels inserum or plasma samples is the preferred method of determining thevitamin D status of a subject.

[0005] 25-hydroxy vitamin D is found circulating in the serum or plasma,tightly bound to DBP (also known as Gc globulin). It is hydrophobic, andin addition to binding to DBP, it is found to bind strongly to otherbinding proteins such as albumin. The presence of such binding proteins,in particular DBP, presents a major difficulty in measuring serum orplasma levels of 25-hydroxy vitamin D.

[0006] To deal with this problem, methods for measurement of 25-hydroxyvitamin D to date have relied upon removal of DBP or other bindingproteins from the sample to be analysed. This has typically involved anextraction step which separates the DBP from the vitamin D metaboliteand removes the DBP from the sample, leaving behind the vitamin Dmetabolite. Extraction has been achieved by a number of methods,including solvent based extraction by adding to the sample an organicsolvent such as chloroform, hexane or ethyl acetate and hexane. Theorganic and aqueous layers are separated and the solvent evaporated. Theresidue is then reconstituted in a water miscible solvent such asethanol. Reverse phase cartridge extraction methods have also been used.Other traditional methods include the use of HPLC and chromatography toachieve separation of individual vitamin D metabolites and exclude fromthe sample interfering factors such as binding proteins.

[0007] Simplification of the methods for extraction and separation hasbeen a key feature in the improvement of assays for vitamin D and itsmetabolites. For example, the chromatography step has been eliminated bythe use of water miscible solvent such as acetonitrile to precipitatebinding proteins. The binding proteins are then separated from thesupernatant by centrifugation, and the supernatant is then analysed byimmunoassay or competitive protein binding assay to determine the amountof vitamin D metabolite present.

[0008] While removal of vitamin D binding proteins from serum samples byorganic solvents is effective, the process requires evaporation ofvolatile organic solvents which is not attractive for routine use inclinical biochemical laboratories. Although the use of water misciblesolvents such as acetonitrile has largely eliminated the need forsolvent evaporation, these have the disadvantage of being toxic. Inaddition, the requirement for a separate extraction step inevitablylengthens the assay time.

[0009] Boullion et al (Clin. Chem. 30/11 1731-1736 (1984)) describe “TwoDirect (Non-Chromatographic) Assays for 25-Hydroxy Vitamin D”. Whilstthe assays described do not require a chromatography step as required inmore traditional methods, they still require extraction of the vitamin Dbinding proteins from the sample by the use of solvent precipitation.Thus, the assays described in this publication suffer from the drawbacksmentioned above.

[0010] Holick et al (U.S. Pat. No. 5,981,779) describe methods forassaying vitamin D and its' metabolites in milk and other biologicalfluids. One of the solid phase assays described uses a competitivebinding assay to measure the vitamin D metabolite in the sample, andcomprises the steps of: (a) providing a solid phase support havingimmobilised thereon a protein or antibody which is capable of bindinglabelled vitamin D; (b) contacting the solid phase with a solution oflabelled vitamin D to allow it to bind to the solid phase; (c) washingthe solid phase to remove unbound labelled vitamin D; (d) contacting thesolid phase with a sample suspected of containing vitamin D or itsmetabolite, for a time sufficient to displace the labelled compound; (e)collecting the liquid of (d); and (f) measuring the amount of labelledvitamin D in the liquid, which is proportional to the amount of vitaminD or metabolite in the test sample. This method is shown as a schematicin FIG. 3, when performed using milk as the test sample.

[0011] The method described is essentially a “free hormone” assay. Inthe example illustrated, the sample is milk which contains little DBP.The hydrophobic vitamin D will be bound to other milk proteins, thebinding of which will be relatively low affinity compared with thebinding for DBP. This is the basis on which a suitable competitionbetween sample-derived vitamin D and labelled vitamin D tracer isachieved in the assay for milk samples. In samples containingsignificant amounts of DBP, for example serum, where the DBP level is˜400 mg/L, approximately 6% of the α-globulin fraction of serum, the“free” (i.e. that not bound to DBP) vitamin D is a very small proportionof the total (0.04% for serum). Such an assay is not suitable formeasurement of vitamin D metabolite in these samples, a view which isfurther supported by Holick et al in a recent publication describing bya variation of the illustrated assay for application to serum samples.However, the only significant difference between the illustrated milkassay and the serum assay is the need for an additional solventextraction stage to remove DBP and other proteins from the sample(11^(th) Workshop on Vitamin D, 27 May-1 Jun. 2000, Nashville, USA). Ifserum was substituted for milk in the original assay, the labelledvitamin D originally bound to the DBP on the tube would simplyequilibrate with the DBP present in the sample, of which 98% haveunoccupied D binding sites. Such an assay would therefore only provide ameasure of the capacity of the sample to bind a vitamin D-analogue, andnot the sample's vitamin D content.

[0012] The Holick patent teaches that measurement of vitamin D (ormetabolites) is possible in milk samples without need for an extractionprocess. It does not teach efficient methods by which the difficultiesof vitamin D measurement in serum can be overcome.

[0013] Measurement of steroid hormones in serum, plasma or otherbiological fluids is carried out by a direct immunoassay, without theneed for a separate extraction procedure. This has been achieved by theuse of a steroid analogue which binds to the relevant steroid bindingproteins, but does not cross react with the antibody used in theimmunoassay. The steroid analogue saturates the steroid binding protein,displacing the steroid and allowing the steroid to bind to theantibodies of the immunoassay.

[0014] Use of a (specific) competitive displacer such as a vitamin Danalogue that does not cross-react with the assay antibody, should beable to provide a “direct assay” (by analogy to direct steroidmeasurement methods). However there are two difficulties. Firstly, theconcentration of DBP is very high in serum samples, typically ˜400 mg/L(˜7 μmol/l), and typically only ˜2% of DBP has 25 hydroxy-vitamin Dbound, the remaining ˜98% DBP remains unoccupied and will bind potentialcompetitive displacers. As a result the concentration of a specificcompetitive displacer must be sufficient to saturate the DBP and wouldneed to be in excess if the affinity was lower than that of 25hydroxyvitamin D. Vitamin D analogues are particularly expensive due tothe complexity of vitamin D chemistry. Secondly, the specificity of DBP(for natural vitamin D metabolites) means that even with theavailability of large numbers of different vitamin D analogues, very fewhave affinity for DBP that can compete with 25 hydroxyvitamin D.

[0015] The present invention aims to overcome or ameliorate the problemsassociated with the prior art, by providing an improved method formeasuring vitamin D metabolite present in a sample of serum or plasma.

[0016] Thus, in a first aspect of the present invention there isprovided a method for measuring vitamin D metabolite present in a plasmaor serum sample of a subject, the method comprising:

[0017] (a) adding to a sample of serum or plasma a non-competitivedisplacement agent to effect separation of any vitamin D metabolite inthe sample from protein to which it is bound, such that any vitamin Dmetabolite can be detected and/or measured; and

[0018] (b) detecting or measuring the amount of vitamin D metabolite inthe sample of (a).

[0019] Thus, the present invention satisfies the pressing need for asimple yet effective method for measuring vitamin D metabolite in aserum or plasma sample. It is based upon the surprising discovery ofnon-competitive displacement agents which enable effective separation ofvitamin D metabolites from binding proteins to enable the amount ofvitamin D metabolites to be detected or measured, without competing withthe protein or requiring its extraction from the sample. In essence, theinvention provides for the first time displacement agents for use invitamin D metabolite analysis which are not analogues or competitors ofthe vitamin D metabolites to be measured. The elimination of the need ofboth the extraction step and competitive displacement agents has enableda method which is more efficient and cost effective than previousmethods, and thus more suited to routine use in clinical biochemicallaboratories or personal use.

[0020] The present invention may be performed on any sample of plasma orserum, preferably from a subject. The subject whose plasma or serum isto be analyzed may be one for whom it is desirable to determine vitaminD status. Preferably, the subject may be a mammal. More preferably, thesubject is a human, and most preferably a child.

[0021] Measuring vitamin D metabolite present in a sample of plasma orserum may include both detecting the presence of any vitamin Dmetabolite in the sample, or more preferably, determining the amount ofvitamin D metabolite present. The amount may be compared with a keydetailing whether the amount represents a deficiency or excess ofvitamin D metabolite.

[0022] Any one or more metabolites of vitamin D may be measured in themethod of the present invention. In a preferred embodiment, a specificvitamin D metabolite is measured in a sample, although it is envisagedthat for some applications it may be preferred to measure two or moretypes of metabolite present. Examples of metabolites include 25-hydroxyvitamin D₂, 25-hydroxy vitamin D₃, 24,25-dihydroxy vitamin D₃ and25,26-dihydroxy vitamin D. 25-hydroxy vitamins D₂ or D₃, or analoguesthereof, are the preferred metabolites to be measured in the method ofthe invention.

[0023] The method of the invention is not restricted to the separation,or displacement, of a vitamin D metabolite from DBP, but may be used inthe separation of a vitamin D metabolite from any factor to which it isbound. Typically, such factors will be proteins, for example, serumalbumin.

[0024] By displacement is meant full or partial separation of some orall of the vitamin D metabolite from the factor to which it is bound inthe sample. Preferably, the displacement of the vitamin D metabolitefrom the binding factor is sufficient to enable the vitamin D metaboliteto detected and/or measured, for example in a binding assay. It ispreferred that substantially all of the vitamin D metabolite present inthe sample is sufficiently displaced from the binding factor, to enableit to be measured. In this context, “substantially” means at least 95%,98% and preferably at least 99% of the vitamin D metabolite isdisplaced. This ensures accuracy of a test based upon the method of theinvention.

[0025] Any non-competitive agent capable of achieving displacement, orseparation, of the vitamin D metabolite from the binding protein may beused in step (a) of the method of the invention. Preferred agents foruse in the present invention are chemical reagents which may act bydisrupting or destroying the bond between vitamin D metabolite andbinding factor. Preferred chemical reagents for use as displacementagents are those which bind to vitamin D binding proteins such asalbumin with sufficient affinity to assist displacement of the vitamin Dmetabolite. In a most preferred embodiment of the present invention, thedisplacement agent is a chemical reagent comprising, preferably in abuffer, 8-anilino-1-napthalenesulphonic acid ammonium salt,3-(acetonylbenzyl)-4-hydroxycoumarin and a water miscible solvent. Anysuitable buffer may be used, for example phosphate buffered saline.Suitable water miscible solvents will be known to persons skilled in theart and include ethanol, acetonitrile, propan-1-ol, propan-2-ol,acetone, dimethylformamide and methyl sulphoxide. The preferred watermiscible solvent is methanol. The suitable concentrations andproportions of the three reagents for producing the displacement agentcan be readily ascertained by persons skilled in the art. Preferredconcentrations of the reagents are 0.5 to 10 g/l, more preferably 1.60g/l 8-anilino-1-napthalenesulponoc acid ammonium salt; 50 to 1000 mg/l,and more preferably 160 mg/l 3-(acetylonylbenzyl)-4-hydroxycoumarin and10 to 300 ml/l, more preferably 160 ml/l water miscible solvent such asmethanol.

[0026] Once the vitamin D metabolite has been displaced from the bindingfactor in the sample, the presence or amount of vitamin D metabolite inthe sample may be determined. Any suitable method may be used for thispurpose, and such methods will be familiar to persons skilled in theart. Examples of suitable methods include binding assays, such ascompetitive or non-competitive binding assays, immunoassays, or directlabeling of unbound vitamin D metabolites.

[0027] The step (b) of measuring the amount of vitamin D metabolite inthe sample may be performed subsequent to, or simultaneously with step(a). Preferably, the step (b) is carried out simultaneously with step(a), thus providing a single step assay or measurement of vitamin Dmetabolite in a serum or plasma sample.

[0028] One of the preferred methods for measuring displaced vitamin Dmetabolite in a sample is by way of a competitive binding assay.Suitable competitive binding assays take various forms, and will be wellknown to persons skilled in the art. A typical competitive binding assaywill comprise contacting a receptor with a labeled form of a ligand anda sample suspected of containing an unlabelled form of the same ligand.The amount of labeled ligand which is found bound to the receptor isindicative of the proportion of unlabeled ligand in the sample.Alternatively, the competitive binding assay may comprise providingreceptors bound to a labeled form of the ligand, adding to the receptorsthe sample suspected of containing the unlabelled form of the ligand,and measuring the amount of displaced labeled ligand which is indicativeof the amount of unlabelled ligand present.

[0029] In a preferred embodiment of the first aspect of the invention,there is provided a method of measuring the amount of displaced vitaminD metabolite in a sample, comprising the steps of:

[0030] (a) providing a support having immobilised thereon a bindingfactor capable of binding a vitamin D metabolite;

[0031] (b) contacting the support with a sample comprising the vitamin Dmetabolite to be measured;

[0032] (c) contacting the support with a labeled form of the vitamin Dmetabolite;

[0033] (d) measuring the amount of labeled vitamin D metabolite leftbound to the support,

[0034] wherein the amount of labeled vitamin D metabolite bound to thesupport is proportional to the amount of vitamin D metabolite in thesample.

[0035] Where it is preferred to perform the binding assay simultaneouslywith the displacement step, the method of the first aspect may comprisethe steps of

[0036] (a) providing a support having immobilised thereon a bindingfactor capsule of binding a vitamin D metabolite;

[0037] (b) contacting the support with a serum or plasma sample of asubject;

[0038] (c) adding to the support a non-competitive displacement agent toeffect separation of the vitamin D metabolite from protein to which itis bound;

[0039] (d) contacting the support with a labelled form of the vitamin Dmetabolite;

[0040] (e) measuring the amount of labelled vitamin D metabolite leftbound to the support;

[0041] wherein the amount of labelled vitamin D metabolite bound to thesupport is proportional to the amount of vitamin D metabolite in thesample.

[0042] The binding factor to be immobilized on the support may be anyfactor capable of binding a labelled or unlabelled form of the vitamin Dmetabolite. The binding factor may a protein, such as albumin or DBP, oran antibody specific for the particular vitamin D metabolite ofinterest. In a most preferred embodiment, the binding factor is anantibody against the vitamin D metabolite. Preferably, the antibody ispolyclonal.

[0043] In an alternative embodiment, the vitamin D metabolite may bemeasured by way of an immunoassay. Any suitable form of immunoassay maybe used, and these will be known to persons skilled in the art. Forexample, the displaced vitamin D may be captured on a support of vitaminD metabolite specific antibodies, and then quantified using a second,labeled vitamin D metabolite specific antibody.

[0044] Any suitable labeling means may be used in the above methods.Suitable labels include enzymatic labels, such as alkaline phosphatase,peroxidase, biotin-streptavidin, fluorescent labels such as flourescinor rhodamine, or chemiluminescent labels such asluminol, acridiumesters, and 1,2-dioxetanes. Also included are combinations of the above,for example avidin-fluorescin, as an alternative for avidin-HRP.

[0045] In a second aspect of the invention, there is provided a methodfor determining the vitamin D status of a subject, the methodcomprising:

[0046] (a) adding to a sample of serum or plasma a non-competitivedisplacement agent to effect separation of any vitamin D metabolite inthe sample from protein to which it is bound, to produce a samplecomprising unbound vitamin D metabolite and protein; and

[0047] (b) measuring the amount of vitamin D metabolite in the sample of(a).

[0048] The vitamin D status of a subject is highly informative, and maybe used to determine the underlying cause of a number of disease statesincluding rickets, and hyper- or hypo-calceamia. Thus, in a preferredembodiment of the second aspect of the invention, the method relates todetermining the cause of disease in a subject, wherein preferably, thesubject is a human and most preferably a child.

[0049] The present invention is performed in vitro, on a sample of serumor plasma removed from a subject. Thus, preferably, the method comprisesthe additional step of first removing a serum or plasma sample from thesubject. The present invention therefore relates to a non-invasivemethod, the results of which may be used in a determining vitamin Dstatus of a subject, and thus the underlying to disease. The method doesnot however provide a result upon which an immediate medical decisionregarding treatment must be made.

[0050] In a third aspect of the present invention, there is provided adisplacement agent as defined in relation to the first aspect.

[0051] In a fourth aspect of the invention, there is provided the use ofan agent of the third aspect. In a method for measuring vitamin Dmetabolite in a sample of serum or plasma, or for determining thevitamin D status of a subject, preferably the displacement agents areused in methods of the first and second aspects of the invention.

[0052] In a fifth aspect of the invention, there is provided a kit foruse in a method of the first or second aspects, comprising adisplacement agent according to the second aspect of the invention. Thekit preferably also comprises a key showing the correlation between theresults of the assay with the amount of vitamin D metabolite present inthe sample. In a further preferred embodiment, a kit may comprise meansfor carrying out step (b) of the first aspect of the invention. Suchmeans may include one or more of a support, labelled vitamin Dmetabolites, antibodies, proteins on labels. The kit preferably willalso comprise instructions for use.

[0053] The preferred embodiments apply to each aspect, mutatis mutandis.

[0054] The present invention will now be described by way of anon-limiting example, with reference to the following figures, in which:

[0055]FIG. 1 shows a typical calibration curve for the direct25-hydroxyvitamin D enzyme immunoassay.

[0056]FIG. 2 shows a correlation of the direct enzyme immunoassay of thepresent invention with Immunodiagnostic Systems Ltds' Gamma B 25-HydroxyVitamin D radioimmunoassay (IDS catalogue number AA-35F1) with 180 serumor plasma patient samples.

[0057]FIG. 3 shows a correlation of the direct enzyme immunoassay withthe Diasorn 25-Hydroxy Vitamin D ¹²⁵I RIA (catalogue number 68100E,Diasorin, Stillwater, Minn., USA) with 55 serum or plasma patientsamples

EXAMPLE Preparation of Anti-vitamin D Coated Microtitre Plates

[0058] Donkey anti-(sheep IgG) serum and sheep anti-vitamin D were bothpurified by sodium sulphate precipitation to give the corresponding IgGfractions. Microtitre plates (Nunc Maxisorp) were coated with donkeyanti(sheep IgG), a purified IgG, 250 μl per well of 30 mg/l IgG in 10 mMphosphate buffered saline (PBS) and incubated overnight at roomtemperature. The coated plates were washed three times with 10 mMphosphate buffered saline, 0.05% Tween 20(PBST). Sheep anti-vitamin IgG,1 ng/ml in PBS, 0.1% Polypep (Sigma), 0.05% sodium azide, 200 μl perwell was added and plates stored at 2-8C. Plates were washed with PBSTbefore use.

Preparation of Vitamin D Biotin

[0059] A solution of 24,25-dihydroxyvitamin D in methanol (200 μl/mg/mL)was treated with an excess of sodium periodate in water for 1 hour atroom temperature and purified by HPLC chromatography (Hypersil C18column 4.6 mm×125 mm, 50% methanol/water, elution gradient of 50% to100% methanol). The eluted fractions containing (25,26,27-nor)-vitaminD-24-aldehyde were treated with excess carboxymethyl oxime (Sigma)methanol for 4 hours, purified by HPLC chromatography (as above) toprovide 24-CMO-vitamin D. The N-hydroxysuccinimide ester was prepared bytreatment of 24-CMO-vitamin D with excess N-hydroxysuccinimide anddicyclohexylcarbodimide in dioxan. The N-hydroxisuccinimide ester of24-CMO-vitamin D and biotin N-hydroxysuccinimide ester (Sigma) wereadded to bisaminopolyethylene glycol (Sigma) in dioxan solution andallowed to react for 3 hours at room temperature. Excess 24-CMO-vitaminD and biotin were removed by dialysis and the vitamin D-biotin conjugatewas stored at −20C.

Vitamin D Displacement Agent

[0060] The preferred formulation of the vitamin D displacement agent isany conventional buffer, such as phosphate buffered saline solutioncontaining 8-anilino-1-napthalenesulphonic acid ammonium salt (0.5 to 10g/l, preferably 1.6 g/l), 3-(α-acetonylbenzyl)-4-hydroxycoumarin (50 to1000 mg/l, preferable 160 mg/l) and methanol (10 to 300 m/l, preferably160 m/l).

Enzymeimmunoassay

[0061] A typical assay procedure is as follows:

[0062] A portion of sample (25 μl serum or plasma) is diluted with 1 mlof vitamin D displacement agent as described above. A portion of thediluted sample (100 μl) is added to the anti-vitamin D antibody coatedmicrotitre plate, followed by addition of a solution (100 μl)of thevitamin D-biotin conjugate, and incubated for 90 minutes at roomtemperature. The plate was washed three times with 10 mM phosphatebuffered saline containing 0.05% Tween20 (PBST). Avidin peroxidaseconjugate (Sigma) diluted 1:2000 in PBST was added and incubated for 30minutes at room temperature, followed by washing three times with PBST.TMB substrate reagent (Moss Inc.) was added and colour allowed todevelop for 30 minutes. After addition of 0.5M HCL to stop the reaction,the absorbance was recorded at 450 nm.

[0063] A calibration curve is prepared with each batch of samples beinganalysed, and the 25-hydroxyvitamin D values for each sample can be readdirectly from the calibration curve using the absorbance value obtainedfor each sample. This can be performed manually from a calibration curveplotted on graph paper, or more usually by suitable data processingsoftware.

[0064] A typical calibration curve for the direct 25-hydroxyvitamin Denzymeimmunoassay is shown in FIG. 1. 25-Hydroxyvitamin D Absorbance(nmol/L) at 450 nm B/Bo (%) 0 2.436 100% 6.8 2.015 83% 14 1.678 69% 271.330 55% 67 0.815 33% 179 0.484 20% 380 0.332 14%

[0065] To demonstrate effectiveness of the direct 25-hydroxyvitamin Denzymeimmunoassay with patient samples, the assay was compared to twocommercially available radioimmunoassays, both of which use anextraction step as part of sample preparation prior to assay. The tworadioimmunoassays are widely used clinically for the quantitativedetermination of 25-hydroxyvitamin D and other metabolites in humanserum or plasma as part of the assessment of vitamin D sufficiency. Thedirect 25D enzymeimmunoassay shows good agreement and correlation withtwo established extraction radioimmunoassys, thus demonstrating theutility of a direct 25-hydroxyvitamin D enzymeimmunoassay for thequantitative determination of 25-hydroxyvitamin D (and othermetabolites) in serum or plasma specimens (FIGS. 2 and 3).

1. A method for measuring vitamin D metabolite present in a plasma orserum sample, the method comprising: (a) adding to a serum or plasmasample a non-competitive displacement agent to effect separation of anyvitamin D metabolite in the sample from protein to which it is bound,such that any vitamin D metabolite may be detected and/or measured; and(b) detecting or measuring the amount of vitamin D metabolite in thesample of (a).
 2. A method for measuring vitamin D metabolite in a serumor plasma sample according to claim 1, wherein the vitamin D metaboliteis 25-hydroxy Vitamin D or an analogue thereof.
 3. A method formeasuring vitamin D metabolite in a serum or plasma sample according toclaim 1 or 2, wherein the non-competitive displacement agent is achemical reagent.
 4. A method for measuring vitamin D metabolite in aserum or plasma sample according to claim 3 wherein the displacementagent comprises 8-anilino-1-napthalenesulphonic acid ammonium salt,3-(acetonylbenzyl)-4-hydroxycoumarin and a water miscible solvent.
 5. Amethod for measuring vitamin D metabolite in a serum or plasma sampleaccording to claim 5, wherein the water miscible solvent is methanol. 6.A method for measuring vitamin D metabolite in a serum or plasma sampleaccording to any one of claims 1 to 5 wherein the amount of vitamin Dmetabolite is measured in a binding assay.
 7. A method for measuringvitamin D metabolite in a serum or plasma sample according to claim 6wherein the binding assay is a competitive binding assay.
 8. A methodfor measuring vitamin D metabolite in a serum or plasma sample accordingto claim 7 wherein the competitive binding assay comprises the steps of(a) providing a support having immobilised thereon a binding factorcapable of binding a vitamin D metabolite; (b) contacting the supportwith a sample comprising the vitamin D metabolite to be measured; (c)contacting the support with a labeled form of the vitamin D metabolite;(d) measuring the amount of labeled vitamin D metabolite left bound tothe support, wherein the amount of labeled vitamin D metabolite bound tothe support is proportional to the amount of vitamin D metabolite in thesample.
 9. A method for measuring vitamin D metabolite in a serum orplasma sample according to claim 8, wherein the binding factorimmobilised on the support is DBP.
 10. A method for measuring vitamin Dmetabolite in a serum or plasma sample according to claim 8, wherein thebinding factor immobilised on the support is an anti-vitamin Dmetabolite antibody.
 11. A method for measuring vitamin D metabolite ina serum or plasma sample according to any one of claims 8 to 10, whereinthe vitamin D metabolite is labeled with biotin, avidin, a fluorescentmolecule, or a chemiluminescent molecule.
 12. A method for measuringvitamin D metabolite in a serum or plasma sample according to claim 6,wherein the binding assay is an immunoassay.
 13. A method fordetermining the vitamin D status of a subject, comprising the methodaccording to any one of claims 1 to
 12. 14. A method for determining thevitamin D status of a subject according to claim 13 comprising theadditional step of first removing a sample from a subject.
 15. Anon-competitive displacement agent according to any one of claims 3 to5.
 16. Use of a non-competitive displacement agent according to claim 15in measuring the vitamin D metabolite in a serum or plasma sample. 17.Use of a non-competitive displacement agent according to claim 15 in amethod according to any one of claims 1 to
 14. 18. A kit for use inmeasuring vitamin D metabolite in a sample comprising a displacementagent according to claim
 15. 19. A kit according to claim 18 furthercomprising a key showing the correlation between the results of themethod and the amount of vitamin D metabolite present in the sample. 20.A kit according to claim 18 or 19 further comprising means for measuringthe vitamin D metabolite present in the sample.
 21. A kit according toany one of claims 18 to 20 further comprising one or more of a support,labels, protein, antibody and instructions for use.